Virus-induced human APOBEC3G gene transcription. (A) Determination of the binding specificity of human APOBEC3F and human APOBEC3G primers in quantitative real time PCR (qRT-PCR). Serial dilutions of the C-terminally HA-tagged plasmids pcDNA_huAPOBEC3F (hA3F) (described by H. Muckenfuss and colleagues) or pcDNA_huAPOBEC3G (generously provided by Nathaniel R. Landau) were analysed using either human APOBEC3F or APOBEC3G specific primer pairs in qRT-PCR. The copy number of each plasmid and the corresponding CT-values (cycle number of the first detectable signal) are given. Low CT-values indicate high amounts of the DNA-sequence of interest. CT-values above 30 are commonly considered as non-specific signals. The qRT-PRC-program is limited to 40 cycles; due to that fact that CT-values >40 indicate undetectable amounts of DNA. (B-E) The influenza virus strain A/Puerto Rico/8/34 H1N1 (PR8) was diluted in PBS containing 0.6% sterile BSA and 1% penicillin/streptomycin and used to infect A549 cells (B and D-E) or HUVEC (C) (MOI = 5) for the time points indicated. At two-hourly intervals post infection, RNA was isolated using the RNeasy mini-kit (Qiagen), and 3 μg of total RNA were transcribed into cDNA using 0.5 μg oligo dT-primer (16 mer) and 200 U Revert Aid H- (Fermentas) according to the manufacture's protocol. mRNA levels of IFNβ, human APOBEC3F and human APOBEC3G were assessed by qRT-PCR using primer pairs for human APOBEC3F and 3G; or for human IFNβ as follows: IFNβ_fwd 5'-GGC CAT GAC CAA CAA GTG TCT CCT CC-3' and IFNβ_rev 5'-GCG CTC AGT TTC GGA GGT AAC CTG T-3'. Induced transcription of mRNA was calculated as n-fold using GAPDH as reference gene. (D) Determination of the copy numbers per cell of human APOBEC3F or human APOBEC3G. (E) Infected A549 cells were lysed at the time points indicated. Endogenous expression of human APOBEC3G was determined with the hA3G specific antibody ApoC17 (NIH AIDS Research and Reference Reagent Program) in Western blots. An anti-tubulin (B5-1-2, Sigma) blot served as a loading control.